Major histocompatibility complex (MHC) class I molecules display peptides derived from endogenous proteins that have been degraded within the cell. Increased cell proliferation and deregulated phosphorylated pathways, which regulate protein activation and degradation in breast cancer cells could lead to a variety of peptide antigens that are different from those present on normal breast epithelial cells. Therefore, we hypothesize that phosphopeptide, phosphate-containing peptides, will be antigens that reflect the processing and presentation of substrates of the kinase pathways active in breast cancer cells. This will therefore make phosphopeptides great candidates for immunotherapy and we wish to explore their use as immunotherapeutic agents for breast cancer patients. The broad long-term goals of this project are to increase the knowledge about breast cancer-associated antigens, reveal information about potential cellular transformation mechanisms, and determine whether the identified antigens can be potential targets for use in the treatment of breast cancer. The specific aims are to (1) identify phosphopeptides that are presented by MHC class I molecules on the cell surface of breast carcinoma cells, which are not presented by the same MHC molecules on normal breast epithelial cells;(2) evaluate the presence of phosphorylation sites of source proteins identified in specific aim I in different types breast carcinomas;and (3) evaluate the immunogenicity of phosphopeptides that are displayed on breast carcinoma cells. Research design: immunoaffinity purification and mass spectrometry will be used to isolate, identify, and sequence HLA- A*0201-restricted phosphopeptides from breast cancer cell lines and normal breast epithelial cell lines. The source proteins of the phosphopeptides will be identified by matching the sequence of the phosphopeptides to protein sequences documented in nonredundant databases. We will employ western blot and immunohistochemistry assays to assess phosphorylation sites of the source proteins in different breast cancer cells not evaluated by mass spectrometry in order to define phosphoprotein expression that are characteristic of different types and/or stages of breast cancer. Proteins common to the breast cancer cells and those that are differentially expressed on the breast cancer cell lines will be evaluated for their ability to activate the immune system. To do this, C57BI/6 AAD transgenic mice will be immunized with synthetic HLA-A*0201-restricted phosphopeptides and the effector function of the CD8+ T cells will be determined by flow cytometry, 51Cr-release, and ELISA assays. We hope to identify new breast cancer antigens which are more effective as immunotherapeutic agents for breast cancer patients.